Escherichia coli has for decades been the most extensively studied bacterium, making it a key model for the living cell and arguably the one about which the most is known. Yet through genome sequencing we have recently become aware of the true level of level of our ignorance. At least half of the genes discovered are simply of unknown function. In this application we propose to systematically characterize these genes by the use of a set of about 5000 PCR amplified DNA fragments- corresponding to each gene. This PCR geneset will be used to carry out global gene expression experiments at the single gene level. That is, for each gene we will determine how its expression is regulated in response to nutritional, physical and genetic stimuli. This is significant because the regulatory circuitry of E. coli is functionally organized. How a gene is regulated gives information about its function. In addition the PCR geneset will be cloned into vectors designed to facilitate additional analyses including mutational, gene inactivation, gene expression and two hybrid system approaches for determining function. Some of these characterizations will be performed as part of this project. For other approaches the PCR clonesets will be made available to interested investigators. Previous work funded under this grant revealed the power of the global gene expression to discover and identify genes in E. coli, even though the technology available at the time permitted resolution only at the 10 gene level. At the single gene level, a decisive advance in understanding of the organism will be possible.